Non-ribosomal peptide synthetases (NRPSs) are multi-domain modular biosynthetic assembly lines that polymerize amino acids into a myriad of biologically active and often structurally complex molecules, including the life-saving non-ribosomal peptide (NRP) antibiotics vancomycin, daptomycin, and penicillin. To increase structural diversity, NRPSs draw from a pool of 23 proteinogenic and about 500 non-proteinogenic amino acids. Terminal thioesterase (TE) domains of modular NRPSs employ diverse release strategies for off-loading thioester-tethered polymeric peptides from termination modules via hydrolysis, aminolysis, reduction, or cyclization to provide mature antibiotics as carboxylic acids, esters, amides, aldehydes, macrolactams, and macrolactones, respectively.
Among the different types of macrolactones, strained β-lactone rings are found in diverse classes of natural products including polyketides (PKs), nonribosomal peptides (NRPs), amino acids, terpenoids, and hybrid molecules. Little is known about the biosynthetic origins of β-lactones despite wide therapeutic value of naturally occurring peptide β-lactones as inhibitors of enzymes in the serine hydrolase superfamily. One naturally occurring peptide β-lactone is tetrahydrolipstatin, a hybrid PK-NRP lipase inhibitor used as a treatment of obesity. Another naturally occurring peptide β-lactone, salinosporamide A (also known as marizomib and NPI-0052) is a hybrid PK-NRP proteasome inhibitor under clinical investigation as a treatment of multiple myeloma and other advanced malignancies. Other naturally-occurring β-lactones are reported to show promise as antimicrobial, anticancer, antiviral, and anti-obesity agents. The genetic and biochemical basis for β-lactone ring formation is an ongoing challenge in natural product biosynthesis, and to date no enzymes are known to catalyze the formation of a strained β-lactone ring.
There is precedent for enzymatic formation of strained 4-membered rings in the closely related β-lactam family of antibiotics. Three chemically distinct biosynthetic pathways leading to β-lactams have been reported. For example, penicillin and cephalosporin bicyclic β-lactam scaffolds may be formed via oxidative cyclization of a NRP tripeptide precursor by isopenicillin N synthase. In another example, the β-lactam rings in clavulanic acid and carbapenems may arise via ATP-dependent cyclization of n-amino acid precursors catalyzed by the β-lactam synthetase enzyme family. In an additional example, the nocardicin family of monocyclic β-lactam antibiotics is derived from NRPS assembly lines with five catalytic modules that each covalently tethers the evolving substrate as a thioester on a peptidyl carrier protein, also known as thiolation (T) domain. In another additional example, a condensation domain of module 5 was identified with a rare HHHXXDG motif that is important for dehydration of a T4-thioester tethered serine to the corresponding dehydroalanine. The newly formed T4-dehydroalanine thioester serves as electrophile for a Michael addition/nucleophilic acyl substitution reaction cascade with the nucleophilic α-amino group of a downstream T5-tyrosine thioester to produce the nocardicin β-lactam warhead. Potentially, modifications of these biosynthetic strategies may be employed to assemble β-lactone rings.
The biosynthetic gene clusters for the β-lactones lipstatin (FIG. 5E), ebelactone (FIG. 5A), salinosporamide (FIG. 5C), and oxazolomycin (FIG. 5B) are known, and corresponding biosynthetic pathways have been previously proposed. The mechanisms for β-lactone ring formation in these systems remain unclear and no enzyme domains have been experimentally linked to β-lactone cyclization. Interestingly, the terminating PKS or NRPS module for each β-lactone antibiotic lacks an embedded thioesterase (TE) domain. TE domains participate in acyl transfers, epimerization of stereogenic centers, proofreading, and release of tethered substrates from the enzymatic assembly line via hydrolysis or macrocyclization.
Lactacystin is a secondary metabolite produced by Streptomyces sp. OM-6519 and is structurally related to salinosporamides. Lactacystin is thought to be cleaved from the biosynthetic assembly line via transthioesterification with N-acetylcystein. The resulting β-hydroxy-N-acteylcysteinylthioester has been shown to be in equilibrium with the cis-fused bicyclic β-lactone believed to be an active proteasome inhibitor. It was previously demonstrated that the trans-monocyclic β-lactone ring found in ebelactone and lactacystin may form non-enzymatically from cyclization of the β-hydroxy-N-acetylcysteamine thioester in aqueous buffer at pH 7. The facile non-enzymatic formation of ebelactone and lactacystin β-lactones from precursor β-hydroxy-thioesters under these conditions may reduce the likelihood of identifying a biosynthetic enzyme catalysis of β-lactone ring formation. However, under certain conditions, enzyme catalysis of β-lactone ring formation may still prove advantageous, despite the relative stability of the β-lactones in aqueous solutions.
Obafluorin in its closed-ring form (RC-Obi) is a cis-monocyclic β-lactone antibiotic (see FIG. 1E) produced by plant-associated strains of P. fluorescens. RC-Obi was discovered during a highly selective antibiotic screening for β-lactam antibiotics that were deactivated in an assay organism expressing β-lactamase resistance enzymes, along with the N-acetyl threonine β-lactone compound SQ26,517. 25 Pseudomonads isolated from diverse soil habitats are known to produce RC-Obi, suggesting a conserved evolutionary role of this molecule in diverse soil ecosystems.
RC-Obi was reported to have broad-spectrum antibacterial activity and demonstrated efficacy in a Streptococcus pneumonia murine septicemia model. Although the biological target of RC-Obi is unknown, bacterial transpeptidases are thought to be potential targets due to the structural similarity of RC-Obi to monocyclic β-lactam antibiotics and the documented susceptibility of RC-Obi to hydrolysis by β-lactamases.
FIG. 1D is a chemical structure diagram illustrating the arrangement of atoms within a 3D model of the RC-Obi structure with color-coded surfaces corresponding to CDSs involved in fragment biosynthesis. The illustrated structure was generated using PyMOL v1.7 software and 3D coordinates obtained from PubChem CID 146354. As illustrated in FIG. 1D, the unique structure of RC-Obi contains a 2,3-dihydroxybenzoic acid (2,3-DHB) unit (pink-shaded region) coupled through an amide linkage (blue-shaded region) to an α-amino-β-lactone ring of the unusual nonproteinogenic amino acid β-hydroxy-p nitro-homoPhenylalanine (β-OH-p-NO2-homoPhe).
FIG. 1E is a schematic diagram illustrating the non-enzymatic hydrolysis of obafluorin β-lactone (RC-Obi) to form a β-hydroxy carboxylic acid, the open-ring form of obafluorin (RO-Obi). Previous studies of the total synthesis of RC-Obi and related analogs confirmed the structure and biological activity of RC-Obi as well as demonstrating that RC-Obi hydrolyzes rapidly in aqueous solutions, as illustrated in FIG. 1E. A previous study using stable isotope feeding to investigate the biosynthesis of RC-Obi indicated that 2,3-DHB and β-OH-p-NO2-homoPhe may be advanced intermediates in the biosynthesis of RC-Obi.